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ThesisItem Open Access Polymerase chain reaction for the detection of canine parvovirus in faeces of dogs(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Josemi, Mathew; KAU; Mini, MA study was undertaken to compare the ability of haemagglutination (HA), polymerase chain reaction (PCR) and seminested PCR for the diagnosis of canine parvovirus (CPV) infection by detecting CPV from faecal samples of clinically suspected dogs. Characterization of vaccine strain and field strains of CPV was performed by restriction enzyme analysis (REA) in this study. Attempts were made to isolate CPV from faecal samples in MDCK cell line. One hundred and twentysix faecal samples were collected from dogs suspected for CPV infection and 40 faecal samples were also collected from normal healthy non-vaccinated as well as vaccinated dogs after 15 days of vaccination, that were brought to veterinary hospitals attached to KAU. All the samples were screened by HA, PCR and seminested PCR to detect CPV. No significant difference in HA titre could be appreciated on comparing the titre using PBS, pH 7.2 and PBS – BSA as diluents. The haemagglutination reaction by CPV was found to be favoured by a slightly acidic pH in the range of 4.0 to 6.0. Chloroform treatment of faecal samples had no influence on HA titres above 5 log2. Among 126 faecal samples screened, 43.65 per cent, 61.11 per cent and 73.81 per cent were tested positive for CPV infection by HA, PCR and seminested PCR respectively. Therefore, seminested PCR was found to be a more sensitive and specific method over HA and PCR for the early diagnosis of CPV infection. All the faecal samples from healthy non-vaccinated and vaccinated dogs after 15 days of vaccination were tested negative by HA, PCR and seminested PCR. Restriction enzyme analysis using HinfI, Rsa I and Sau 961 revealed no difference in the fragment length patterns between CPV vaccine strain and field strain. Attempts to isolate CPV from faecal samples in MDCK cell line were found unsuccessful. The occurrence of CPV infection among non-vaccinated dogs was found to be higher than that of vaccinated dogs. Vaccine failure was also observed after MLV vaccination. The distribution of CPV infection was highest among dogs between two to four months of age. Breed-wise distribution of CPV infection showed highest distribution in German shepherd dogs when compared to other breeds. Most of the cases of CPV were noticed during February and July in the year 2003 and during March to June in the year 2004.ThesisItem Open Access Role of newcastle disease, infectious bronchitis and egg drop syndrome-76 viruses in low egg production of chicken in Kerala(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2004) Arun, A; KAU; Mini, MThe present study has been undertaken to assess the prevalence of Newcastle disease (ND), infectious bronchitis (IB) and egg drop syndrome-76 (EDS-76) viruses in apparently healthy layer chicken in Kerala by seroprevalence studies and virus isolation trials and to associate these viruses with low egg production. Sera, cloacal and tracheal swab samples were collected from two to five per cent of total population of a farm to carry out the study. Samples were collected from Regional Poultry Farms (RPF) of Kerala, University Poultry Farm, Mannuthy and also from birds brought to the department of microbiology for disease investigation. To assess the production performance of the layers data pertaining to the egg production were collected, for the past one to five years depending upon the availability. The haemagglutination inhibition (HI) test was used to study the seroprevalence of ND. Out of 615 samples tested, 548 samples were found to be positive. Newcastle disease vaccination was carried out regularly in all the organized farms and the titre is to be considered as an effect of vaccination. Seven virus isolates were recovered from cloacal swab samples and was identified as NDV by HI test. The isolates were subjected to conventional characterization by mean death time, intracerebral pathogenicity index, intravenous pathogenicity index, and stability of haemagglutinins at 56º C, agglutination of mammalian erythrocytes, and adsorption of haemagglutinins by chick embryo brain cells. Results of these tests identified all the seven isolates as lentogenic. The isolates produced mild cytopathic effects (CPE) like syncytia, pyknosis of nuclei and vacuolation of nuclei in chick embryo fibroblast (CEF) monolayer culture. Immunofluorescence test (IFT) was carried out in CEF monolayer using NDV antiserum and specific fluorescence was noticed in the cytoplasm. All the isolates were subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for amplifying 254 bp fusion protein cleavage site (FPCS) sequence gene. Sera samples were tested by ELISA for detecting IB antibodies due to the erratic results of HI test. Out of 418 sera samples screened, 311 (74.40 per cent) samples were found to be positive for IB. Since IB vaccination was not followed in any of the farms in Kerala, the antibodies might be due to past/subclinical infection. One IBV isolate was recovered from tissue samples of ailing broiler bird and was identified by AGID test. The ciliostatic effect of IBV was studied in tracheal organ culture (TOC) and the virus was identified in TOC by IFT and immunoperoxidase test (IPT). The IB virus was confirmed by RT-PCR using primers for a 464 bp nucleotide sequence of S1gene. The antibodies against EDS-76 virus were identified by HI test. Out of 615 chicken sera samples tested, 58 (9.43 per cent) were found to be positive. None of the swab and tissue samples yielded EDS-76 virus. By correlating the production performance of chicken with seroprevalence and isolation trials ND, IB and EDS-76 viruses, this study revealed the possible involvement of IB and EDS-76 viruses in low egg production of chicken in Kerala, because the birds are well protected against ND by vaccination.ThesisItem Open Access Assessment of different experimental vaccines against Chlamydophila abortus (Chlamydia psittaci)in rabbits(Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy, 2003) Sanjeetha, L; KAU; Mini, MTwo isolates of Chlamydia psittaci viz., M-430 and M-28, maintained in the Department of Microbiology, were used in the study. M-430 was used for the preparation of inactivated yolk sac and elementary body vaccines and M-28 was used for challenge experiment. These isolates were revived by inoculating to six to eight day-old chicken embryo through yolk sac route. Both isolates produced characteristic lesions in the embryo and yolk sac membrane. M-430 was also propagated in Mc Coy cell line for high yield of elementary bodies for vaccine preparation. Homogenous suspensions of the yolk sac and elementary body vaccines were inactivated with formalin to a final concentration of 0.4 per cent. Pure and safe preparations were used for vaccination trial. Immunogenic potential of the vaccines were tested in rabbits by giving two doses of each of the vaccines. The first dose was given at three months of age and second dose was given 14 days after the first. Three vaccinated and control rabbits were challenged with M-28 isolate on 28th day post vaccination and rest three on 70th day post vaccination. The clearance of elementary bodies from the tissues (lung, liver lymphnode and spleen) of vaccinated rabbits was an indication of the protection conferred by the vaccines. Better response was noticed with EB vaccine than yolk sac vaccine. The sera were collected from rabbits at regular intervals of 0, 7, 14, 21, 28, 42, 56 and 70 for passive haemagglutination and serum neutralization test. Both vaccines elicited good immune response. The greater humoral immune response of the rabbits that received EB vaccine suggests its slight superiority over the yolk sac vaccine. More evaluation and elaborated field trials on target species are required before advocating the vaccine for field use